Anti-pd-l1 monoclonal antibodies and fusion proteins with interleukin-15, interleukin-15 receptor alpha, or interleukin-2

Abstract

The instant disclosure provides proteins with antibody heavy chain variable domains and light chain variable domains that can be paired to form antigen-binding sites that specifically bind to PD-L1. In certain embodiments, the proteins or antigen-binding sites form antibody or bispecific antibody, such as, for example, PD-L1/IL-2Rβ bispecific antibody. Also provided are pharmaceutical compositions comprising such proteins, therapeutic methods for using such proteins, and pharmaceutical compositions thereof, including for the treatment of cancer.

1 . An antigen-binding site that binds PD-L1, comprising:
 (a) a heavy chain variable region (VH) comprising a complementarity determining region 1 (CDR1) sequence, a complementarity determining region 2 (CDR2) sequence, and a complementarity determining region (CDR3) sequence of SEQ ID NOS: 11, 12, and 89, respectively; and a light chain variable region (VL) comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NOS: 47, 48, and 49, respectively   (b) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 3, 4, and 5, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 6, 7, and 8, respectively;   (c) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 13, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 14, 15, and 16, respectively;   (d) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 19, 20, and 21, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 22, 23, and 24, respectively;   (e) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 19, 20, and 27, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 28, 29, and 30, respectively;   (f) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 33, 34, and 35, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 36, 37, and 38, respectively;   (g) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 3, 41, and 5, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 6, 42, and 43, respectively;   (h) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 46, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively;   (i) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 52, 53, and 54, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 55, 56, and 57, respectively;   (j) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 46, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 60, and 49, respectively;   (k) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 63, 64, and 65, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 66, 67, and 68, respectively;   (l) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 71, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively;   (m) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 74, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively;   (n) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 77, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively;   (o) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 80, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively;   (p) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 83, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively; or   (q) VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 11, 12, and 86, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOS: 47, 48, and 49, respectively.   
     
     
         2 . (canceled) 
     
     
         3 . The antigen-binding site of  claim 1 , wherein:
 (a) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 87 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 88;   (b) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2;   (c) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10;   (d) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 18;   (e) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 26;   (f) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 31 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 32;   (g) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 39 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 40;   (h) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45;   (i) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 50 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 51;   (j) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 58 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 59;   (k) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 62;   (l) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 69 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 70;   (m) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 72 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 73;   (n) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 75 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 76;   (o) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 78 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 79;   (p) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 81 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 82;   (q) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 84 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85;   (r) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 87 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:88   (s) the VH comprises or consists of the amino acid sequence of SEQ ID NO:1 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:2;   (t) the VH comprises or consists of the amino acid sequence of SEQ ID NO:9 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:10;   (u) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 17 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:18;   (v) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO:26;   (w) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 31 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:32;   (x) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 39 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:40;   (y) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:45;   (z) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 50 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:51;   (aa) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 58 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:59;   (bb) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 61 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:62;   (cc) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 69 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:70;   (dd) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 72 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:73;   (ee) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 75 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:76;   (ff) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 78 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:79;   (gg) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:82; or   (hh) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 84 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:85.   
     
     
         4 . (canceled) 
     
     
         5 . The antigen-binding site of  claim 1 , wherein the antigen-binding site is a single-chain fragment variable fragment (scFv) or antigen-binding fragment (Fab). 
     
     
         6 . (canceled) 
     
     
         7 . The antigen-binding site of  claim 1 , wherein:
 (a) the antigen-binding site binds human PD-L1 with an KD less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.8 nM, less than about 0.6 nM, less than about 0.4 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by surface plasmon resonance (SPR);   (b) the antigen-binding site binds human PD-L1 with an EC50 less than about 160 nM, less than about 10 nM, less than about 1.5 nM, less than about 1.2 nM, less than about 1.0 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, or less than about 0.4 nM, as measured by enzyme-linked immunosorbent assay (ELISA);   (c) the antigen-binding site binds cells expressing human PD-L1 with an EC50 less than about 40 nM, less than about 10 nM, less than about 8 nM, less than about 6 nM, less than about 4 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.3 nM, less than about 0.2 nM, less than about 0.1 nM, as measured by fluorescence-activated cell sorting (FACS); and/or   (d) wherein the antigen-binding site competes with PD-1 for binding PD-L1 or inhibits PD-L1 binding to PD-1.   
     
     
         8 - 10 . (canceled) 
     
     
         11 . A protein comprising the antigen-binding site of  claim 1 , optionally wherein the protein comprises one or more antibody heavy chain constant regions, further optionally wherein:
 (a) the antibody heavy chain constant region is human IgG heavy chain constant region;   (b) the antibody heavy chain constant region is human IgG1 heavy chain constant region;   (c) the antibody heavy chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:90;   (d) the antibody heavy chain constant region comprises, relative to SEQ ID NO: 90, one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V, numbered according to the EU numbering system;   (e) the antibody heavy chain constant region comprises an amino acid sequence of SEQ ID NO:91 or SEQ ID NO:92; and/or   (f) the protein comprises a first antibody heavy chain constant region comprising, relative to SEQ ID NO:90, one or more mutations selected from S354C, T366W and K409A and a second antibody heavy chain constant region comprising, relative to SEQ ID NO:90, one or more mutations selected from Y349C, T366S, L368A, F405K and Y407V, numbered according to the EU numbering system.   
     
     
         12 - 18 . (canceled) 
     
     
         19 . The protein of  claim 11 , wherein the protein is an antibody, optionally wherein the antibody comprises:
 (a) a heavy chain (HC) comprising an amino acid sequence at least 90% identical to SEQ ID NO:148 and a light chain (LC) comprising an amino acid sequence at least 90% identical to SEQ ID NO:149;   (b) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 150 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 151;   (c) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 152 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 153;   (d) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 154 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 155;   (e) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 156 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 157;   (f) a HC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 159 and a LC comprising an amino acid sequence at least 90% identical to SEQ ID NO: 160;   (g) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 148 and a LC comprising or consists of the amino acid sequence of SEQ ID NO: 149;   (h) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 150 and a LC comprising or consists of the amino acid sequence of SEQ ID NO:151;   (i) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 152 and a LC comprising or consists of the amino acid sequence of SEQ ID NO:153;   (j) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 154 and a LC comprising or consists of the amino acid sequence of SEQ ID NO:155;   (k) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 156 and a LC comprising or consists of the amino acid sequence of SEQ ID NO: 157; or   (l) a HC comprising or consists of the amino acid sequence of SEQ ID NO: 159 and a LC comprising or consists of the amino acid sequence of SEQ ID NO:160.   
     
     
         20 - 21 . (canceled) 
     
     
         22 . The protein of  claim 19 , wherein;
 (a) the antibody binds human PD-L1 with an KD of less than about 10 nM, less than about 6 nM, less than about 3 nM, less than about 1 nM, or less than about 0.4 nM, as measured by SPR;   (b) the antibody binds human PD-L1 with an EC50 of less than about 0.2 nM, as measured by ELISA;   (c) the antibody binds cells expressing human PD-L1 with an EC50 of less than about 7 nM, as measured by FACS;   (d) the antibody inhibits tumor growth in vivo; and/or   (e) the antibody induces IFNγ and TNFα secretion in vivo, at a comparable to or more enhanced level as compared to atezolizumab.   
     
     
         23 - 26 . (canceled) 
     
     
         27 . A bifunctional protein, comprising:
 (a) an antigen-binding site that binds PD-L1, comprising:
 (i) a heavy chain variable domain (VH) comprising complementarity-determining region 1 (CDR1) of SEQ ID NO: 11, 3, 19, 33, 52, or 63; complementarity-determining region 2 (CDR2) of SEQ ID NO: 12, 4, 20, 34, 41, 53, or 64; and complementarity-determining region 3 (CDR3) of SEQ ID NO: 89, 5, 13, 21, 27, 35, 46, 54, 65, 71, 74, 77, 80, 83, or 86; and 
 (ii) a light chain variable domain (VL) comprising CDR1 sequence of SEQ ID NO: 47, 6, 14, 22, 28, 36, 55, or 66; CDR2 sequence of SEQ ID NO: 48, 7, 15, 23, 29, 37, 42, 56, 60, or 67; and CDR3 sequences of SEQ ID NO: 49, 8, 16, 24, 30, 38, 43, 57, or 68; and 
   (b) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Rα) polypeptide, a wild-type interleukin-2 (IL-2) polypeptide, or an engineered IL-2 polypeptide, or a functional fragment or variant thereof, optionally further comprising one or more antibody heavy chain constant regions.   
     
     
         28 . (canceled) 
     
     
         29 . The bifunctional protein of  claim 27 , wherein the antigen-binding site that binds PD-L1 comprises:
 (a) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 87 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 88;   (b) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2;   (c) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10;   (d) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 18;   (e) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 26;   (f) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 31 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 32;   (g) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 39 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 40;   (h) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 44 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45;   (i) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 50 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 51;   (j) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 58 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 59;   (k) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 62;   (l) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 69 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 70;   (m) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 72 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 73;   (n) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 75 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 76;   (o) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 78 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 79;   (p) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 81 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 82;   (q) the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 84 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85   (r) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 87 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:88   (s) the VH comprises or consists of the amino acid sequence of SEQ ID NO:1 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:2;   (t) the VH comprises or consists of the amino acid sequence of SEQ ID NO:9 and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 10;   (u) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 17 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:18;   (v) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 25 and the VL comprises the amino acid sequence of SEQ ID NO:26;   (w) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 31 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:32;   (x) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 39 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:40;   (y) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 44 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:45;   (z) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 50 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:51;   (aa) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 58 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:59;   (bb) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 61 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:62;   (cc) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 69 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:70;   (dd) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 72 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:73;   (ee) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 75 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:76;   ff) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 78 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:79;   (gg) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:82; or   (hh) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 84 and the VL comprises or consists of the amino acid sequence of SEQ ID NO:85.   
     
     
         30 . (canceled) 
     
     
         31 . The bifunctional protein of  claim 27 , comprising:
 (a) a first subunit comprising the antigen-binding site that binds PD-L1, an interleukin-15 (IL-15) polypeptide or a functional fragment or variant thereof, and a first antibody heavy chain constant region; and   (b) a second subunit comprising the antigen-binding site that binds PD-L1, an interleukin-15 receptor alpha (IL-15Rα) polypeptide or functional fragment or variant thereof, and a second antibody heavy chain constant region.   
     
     
         32 . The bifunctional protein of  claim 27 , wherein:
 (a) the IL-15 polypeptide comprises an amino acids 50-162 of SEQ ID NO:93 or a functional fragment or variant thereof; and/or   (b) the IL-15Rα polypeptide comprises an amino acids 31-97 of SEQ ID NO: 94 or a functional fragment or variant thereof.   
     
     
         33 . (canceled) 
     
     
         34 . The bifunctional protein of  claim 27 , wherein:
 (a) the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:186 and the second subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:187; optionally wherein the first subunit further comprises an amino acid sequence at least 90% identical to SEQ ID NO:181, optionally wherein the first subunit comprises an amino acid sequence of SEQ ID NO:186 and the second subunit comprises an amino acid sequence of SEQ ID NO:187; optionally wherein the first subunit further comprises an amino acid sequence of SEQ ID NO: 181; or   (b) the first subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO:188 and the second subunit comprising an amino acid sequence at least 90% identical to SEQ ID NO:189, optionally wherein the first subunit comprises or consists of the amino acid sequence of SEQ ID NO:188, and the second subunit comprises or consists of the amino acid sequence of SEQ ID NO: 189.   
     
     
         35 - 37 . (canceled) 
     
     
         38 . The bifunctional protein of  claim 27 , wherein the engineered IL-2 polypeptide comprises:
 (a) an IL-2 receptor a (IL-2Rα) binding region 1 comprising, relative to wild-type IL-2, one or more mutations at one or more positions selected from:   a mutation at position K35 selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C;   a mutation at position R38 selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, and R38N;   a mutation at position F42 selected from F42A, F42R, F42G, F42I, F42L, F42P and F42H; and   a mutation at position Y45 selected from Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F, and/or   (b) an IL-2 receptor β (IL-2Rβ) binding region 2 motif comprising:   
       
         
           
                 
               
                   (SEQ ID NO: 95) 
                 
                   X1-X2-X3-D-X4-X-5-X6-N-X7-X8-X9-X10-X11-X12-X13, 
                 
             
                
                
               
            
           
         
       
       wherein:
 X1 is selected from C, T, G, W, I, S, E, and K; 
 X2 is selected from Y, P, V, W, L, A, and G; 
 X3 is selected from S, T, Q, G, M, E, R, and K; 
 X4 is selected from A, V, S, and T; 
 X5 is selected from I, L, T, and V; 
 X6 is selected from S, T, E, D, and R; 
 X7 is selected from I, A, M, and V; 
 X8 is selected from S, T, N, Q, I, G, E, K, and R; 
 X9 is selected from V, L, and I; 
 X10 is selected from N, T, I, and L; 
 X11 is selected from V, A, and I; 
 X12 is selected from and Q, L, G, K, and R; and 
 X13 is selected from A, D, and E. 
 
     
     
         39 . The bifunctional protein of  claim 38 , wherein the engineered IL-2 polypeptide comprises:
 (a) an IL-2Rα binding region 1 comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 124-147, optionally wherein the IL-2Rα binding region 1 comprises an amino acid sequence selected from SEQ ID NOS: 124-147; and/or   (b) an IL-2Rβ binding region 2 motif comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 96-123, optionally wherein the IL-2Rβ binding region 2 motif comprises an amino acid sequence selected from SEQ ID NOS: 96-123.   
     
     
         40 . (canceled) 
     
     
         41 . The bifunctional protein of  claim 27 , comprising:
 (a) an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 160-164, or   (b) an amino acid sequence selected from SEQ ID NOS: 160-164.   
     
     
         42 . (canceled) 
     
     
         43 . The bifunctional protein of  claim 27 , comprising:
 (a) a first subunit comprising the antigen-binding site that binds PD-L1 and a first antibody heavy chain constant region; and   (b) a second subunit comprising a wild-type interleukin-2 (IL-2) or an engineered IL-2 polypeptide or a functional fragment or variant thereof and a second antibody heavy chain constant region, optionally wherein:
 (i) the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:176 and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 165-175, further optionally wherein the first subunit comprises an amino acid sequence of SEQ ID NO: 176 and the second subunit comprises an amino acid sequence selected from SEQ ID NOS: 165-175; or 
 (ii) the first subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 177-180 and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 165-175, optionally wherein the first subunit further comprises an amino acid sequence at least 90% identical to SEQ ID NO: 181, 
   further optionally wherein:
 (A) the first subunit comprises an amino acid sequence selected from SEQ ID NOS: 177-180 and the second subunit comprises an amino acid sequence selected from SEQ ID NOS: 165-175, optionally wherein the first subunit further comprises an amino acid sequence of SEQ ID NO: 181; or 
 (B) the first subunit comprises the amino acid sequence of SEQ ID NO: 177 and SEQ ID NO:181, and the second subunit comprises the amino acid sequence of SEQ ID NO:171. 
   
     
     
         44 - 48 . (canceled) 
     
     
         49 . The bifunctional protein of  claim 27 , comprising:
 (a) a first subunit comprising the antigen-binding site that binds PD-L1 and a first antibody heavy chain constant region; and   (b) a second subunit comprising the antigen-binding site that binds PD-L1, a wild-type interleukin-2 (IL-2) or an engineered IL-2 polypeptide or a functional fragment or variant thereof, and a second antibody heavy chain constant region   wherein optionally, the first subunit comprises an amino acid sequence at least 90% identical to SEQ ID NO:182 and the second subunit comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOS: 183-185; optionally wherein the first and the second subunit, each independently, further comprises an amino acid sequence at least 90% identical to SEQ ID NO:181, wherein further optionally the first subunit comprises an amino acid sequence of SEQ ID NO:182 and the second subunit comprises an amino acid sequence selected from SEQ ID NOS: 183-185; optionally wherein the first and the second subunit, each independently, further comprises an amino acid sequence of SEQ ID NO: 181.   
     
     
         50 - 51 . (canceled) 
     
     
         52 . The bifunctional protein of  claim 27  comprising one or more antibody heavy chain constant regions, wherein the antibody heavy chain constant region is human IgG heavy chain constant region, optionally wherein:
 (a) the antibody heavy chain constant region is human IgG1 heavy chain constant region; 
 (b) the antibody heavy chain constant region comprises an amino acid sequence of at least 90% identity to SEQ ID NO:90; 
 (c) the antibody heavy chain constant region comprises, relative to SEQ ID NO: 90, one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V, numbered according to the EU numbering system; 
 (d) one of the first and the second antibody heavy chain constant regions comprises, relative to SEQ ID NO:90, one or more mutations selected from S354C, T366W and K409A; and the other antibody heavy chain constant region comprises, relative to SEQ ID NO:90, one or more mutations selected from Y349C, T366S, L368A, F405K and Y407V, numbered according to the EU numbering system; 
 (e) one of the first and the second antibody heavy chain constant regions comprises, relative to SEQ ID NO:90, mutations S354C, T366W and K409A; and the other antibody heavy chain constant region comprises, relative to SEQ ID NO:90, mutations Y349C, T366S, L368A, F405K and Y407V, numbered according to the EU numbering system; and/or 
 (f) one of the first and the second antibody heavy chain constant regions comprises an amino acid sequence of SEQ ID NO:91 and the other antibody heavy chain constant region comprises an amino acid sequence of SEQ ID NO:92. 
 
     
     
         53 - 58 . (canceled) 
     
     
         59 . The bifunctional protein of  claim 27 , wherein:
 (a) the bifunctional protein binds human PD-L1 with a KD of less than about 1 nM or with a comparable or lower KD as compared to the comprised antigen-binding site that binds PD-L1, as measured by SPR;   (b) the bifunctional protein binds PD-L1 with a KD of less than about 0.5 nM or less than about 0.1 nM, as measured by SPR;   (c) the bifunctional protein binds PD-L1 with a EC50 of less than about 0.4 nM, as measured by ELISA;   (d) the bifunctional protein competes with PD-1 for binding PD-L1 or inhibits PD-L1 binding to PD-1; and/or   (e) the bifunctional protein inhibits PD-L1 binding to PD-1.   
     
     
         60 - 63 . (canceled) 
     
     
         64 . The bifunctional protein of  claim 27 , wherein:
 (a) the bifunctional protein binds IL-2Rβ with a KD of less than about 100 nM, less than about 80 nM, less than about 50 nM, less than about 10 nM, or less than about 5 nM, as measured by SPR,   (b) the bifunctional protein binds IL-2Rβ with a KD of less than about 65 nM or less than about 50 nM, as measured by SPR;   (c) the bifunctional protein binds IL-2Rα with a KD of less than about 40 nM, as measured by SPR;   (d) the bifunctional protein binds IL-2Rα with a EC50 of less than about 1 nM, as measured by ELISA; and/or   (e) the bifunctional protein binds IL-2Rβ with a EC50 of less than about 5 nM, less than about 2.5 nM, less than about 1.5 nM, less than about 1 nM, or less than about 0.6 nM, as measured by ELISA.   
     
     
         65 - 68 . (canceled) 
     
     
         69 . The bifunctional protein of  claim 27 , wherein:
 (a) the bifunctional protein induces p-STAT5 expression in immune cells;   (b) the bifunctional protein induces p-STAT5 expression in immune cells with EC50 of less than about 1 nM, less than about 0.6 nM, or less than about 0.1 nM, as measured in isolated human peripheral blood mononuclear cells (PBMCs), and wherein the immune cells are T cells, NK cells, or Tregs;   (c) the bifunctional protein induces p-STAT5 expression in immune cells with EC50 of less than about 0.5 nM or less than about 0.1 nM, as measured in isolated PBMCs, and wherein the immune cells are T cells, NK cells, or Tregs;   (d) the bifunctional protein induces p-STAT5 expression in immune cells as measured in mouse splenocytes, and wherein the immune cells are T cells, NK cells, or Tregs;   (e) the bifunctional protein inhibits tumor growth in vivo; and/or   (f) the bifunctional protein induces immune cell proliferation in vivo, optionally wherein the immune cell is T cell or NK cell, further optionally wherein the T cell is CD8+ T cell.   
     
     
         70 - 76 . (canceled) 
     
     
         77 . An antibody, comprising a VH and VL, wherein the VH comprises the polypeptide sequence of SEQ ID NO:87 and the VH comprises the polypeptide sequence of SEQ ID NO:88, optionally wherein the antibody comprises the polypeptide sequences of SEQ ID NO: 177 and SEQ ID NO: 181. 
     
     
         78 . (canceled) 
     
     
         79 . An isolated polynucleotide encoding an antigen-binding site of  claim 1 . 
     
     
         80 . An expression vector comprising the polynucleotide of  claim 79 . 
     
     
         81 . A modified cell comprising the isolated polynucleotide of  claim 79 . 
     
     
         82 . A pharmaceutical composition comprising the an antigen-binding site of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         83 - 88 . (canceled) 
     
     
         89 . A method of modulating an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antigen-binding site of  claim 1  or a pharmaceutical composition thereof, optionally wherein the modulating the immune response comprises at least one of enhancing T cell activity or enhancing NK cell activity, further optionally wherein the subject is treated with an additional therapeutic agent. 
     
     
         90 . (canceled) 
     
     
         91 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antigen-binding site of  claim 1  or a pharmaceutical composition thereof, optionally wherein the cancer comprises breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, or melanoma. 
     
     
         92 - 93 . (canceled)